*Protein ID (NP or XP #) or Wolbachia#: AHH27801 *Organism (including strain): Klebsiella pneumoniae Etiologic Risk Group: 2 */Disease Information: Klebsiella pneumoniae is a bacterial species that frequently causes respiratory infections such as pneumonia but can also cause soft tissue infections in deadlier cases. Though K. pneumoniae is found in nature and can cause infection anywhere in nature, K. pneumoniae infection cases have increased steadily in hospitals, granting K. pneumoniae infections the title of “nosocomial infection” or hospital-originated infection [1]. Unfortunately, the reason for the increasing number of infections is due to the discovery of antibiotic resistance in recent strains of K. pneumoniaein the United States since 1996 [2]. Prior to 1996, beta-lactam drugs such as penicillin were sufficient to decrease infection. However, the evolution of beta-lactam resistant K. pneumoniae exacerbated the situation for infected patients. These new strains produce enzymes called beta-lactamases that hydrolyze beta-lactam antibiotics. Extremely broad spectrum antibiotics called carbapenems were synthesized and are used as a last-ditch effort to save a patient undergoing a deadly infection. Unfortunately, K. pneumoniae was discovered to have evolved to produce KPC-2, a carbapenamase that hydrolyzes carbapenems and leaves infected patients to suffer without any effective antibiotic [3].A solution to the antibiotic resistance of K. pneumoniae is to discover other chemical compounds that can inhibit carbapenamase and effectively prevent K. pneumoniae from being resistant to currently-available carbapenem antibiotics.
Link to TDR Targets page: None Essentiality of this protein: Essential if KPC-2 is inactive with antibiotics in the environment of K. pneumoniae. Is it a monomer or multimer as biological unit? Monomer Complex of proteins?: No. Druggable Target: K. pneumoniae
Current Inhibitors: Clavulanic acid, tazobactam, penem 1 & 2, imipenem
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method: Ni-NTA with 6xHis tag
Image of protein (PyMol with features delineated and shown separately):
Fig 2: X-ray crystal structure of KPC-2 (PDB: 2OV5). Active site shown as surface at 40% transparency; non-active site residues shown as cartoon; bicine molecule shown as sticks (carbons in cyan, oxygens in red, and nitrogens in blue). Protein coloration: carbons in green, oxygen in red, nitrogen in blue). Polar contacts shown as black dashes.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Primer overlap PCR was not done for KPC-2. Gene obtained from Fast lab in pET24-a vector.
*GC% Content for gene (codon optimized): n/a
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): n/a
*NCBI Gene # or RefSeq#: KP987218
*Protein ID (NP or XP #) or Wolbachia#: AHH27801
*Organism (including strain): Klebsiella pneumoniae
Etiologic Risk Group: 2
*/Disease Information:
Klebsiella pneumoniae is a bacterial species that frequently causes respiratory infections such as pneumonia but can also cause soft tissue infections in deadlier cases. Though K. pneumoniae is found in nature and can cause infection anywhere in nature, K. pneumoniae infection cases have increased steadily in hospitals, granting K. pneumoniae infections the title of “nosocomial infection” or hospital-originated infection [1]. Unfortunately, the reason for the increasing number of infections is due to the discovery of antibiotic resistance in recent strains of K. pneumoniae in the United States since 1996 [2]. Prior to 1996, beta-lactam drugs such as penicillin were sufficient to decrease infection. However, the evolution of beta-lactam resistant K. pneumoniae exacerbated the situation for infected patients. These new strains produce enzymes called beta-lactamases that hydrolyze beta-lactam antibiotics. Extremely broad spectrum antibiotics called carbapenems were synthesized and are used as a last-ditch effort to save a patient undergoing a deadly infection. Unfortunately, K. pneumoniae was discovered to have evolved to produce KPC-2, a carbapenamase that hydrolyzes carbapenems and leaves infected patients to suffer without any effective antibiotic [3].A solution to the antibiotic resistance of K. pneumoniae is to discover other chemical compounds that can inhibit carbapenamase and effectively prevent K. pneumoniae from being resistant to currently-available carbapenem antibiotics.
Link to TDR Targets page: None
Essentiality of this protein: Essential if KPC-2 is inactive with antibiotics in the environment of K. pneumoniae.
Is it a monomer or multimer as biological unit? Monomer
Complex of proteins?: No.
Druggable Target: K. pneumoniae
*EC#: 3.5.2.6
Link to BRENDA EC# page: http://www.brenda-enzymes.info/enzyme.php?ecno=3.5.2.6
Enzyme Assay information:
http://link.springer.com/protocol/10.1007%2F978-1-59745-246-5_19
Structure: PDB: 2OV5
Current Inhibitors: Clavulanic acid, tazobactam, penem 1 & 2, imipenem
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method: Ni-NTA with 6xHis tag
Image of protein (PyMol with features delineated and shown separately):
Fig 2: X-ray crystal structure of KPC-2 (PDB: 2OV5). Active site shown as surface at 40% transparency; non-active site residues shown as cartoon; bicine molecule shown as sticks (carbons in cyan, oxygens in red, and nitrogens in blue). Protein coloration: carbons in green, oxygen in red, nitrogen in blue). Polar contacts shown as black dashes.
*Amino Acid Sequence:
MAEPFAKLEQDFGGSIGVYAMDTGSGATVSYRAEERFPLCSSFKGFLAAAVLARSQQQAGLLDTPIRYGKNALVPWSPISEKYL
TTGMTVAELSAAAVQYSDNAAANLLLKELGGPAGLTAFMRSIGDTTFRLDRWELELNSAIPGDARDTSSPRAVTESLQKLTLGS
ALAAPQRQQFVDWLKGNTTGNHRIRAAVPADWAVGDKTGTCGVYGTANDYAVVWPTGRAPIVLAVYTRAPNKDDKHSEAVIAAA
ARLALEGLGV*
*length of your protein in Amino Acids: 263 aa
Molecular Weight of your protein in kiloDaltons: 27.768 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 39420
TMpred graph Image:
*CDS Gene Sequence:
ATGGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGA
GGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTT
ACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAACAGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGT
GATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCGTCTGGACCG
CTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAAAACTGACACTGGGCTCTG
CACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGCAGTC
GGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCG
GGCGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGACTCGCGCTCGAGGGATTGGGCGTCTAACAGTAAAGGTGGATA
*GC% Content for gene: 61.3%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Primer overlap PCR was not done for KPC-2. Gene obtained from Fast lab in pET24-a vector.
*GC% Content for gene (codon optimized): n/a
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): n/a
Forward:
5’ TACTTCCAATCCATGGCGGAACCATTCGCTAAACT 3’
Reverse:
5’ TATCCACCTTTACTGTTAGACGCCCAATCCCTCGAG 3’